DNA double-strand breaks (DSB)s are induced by a variety of genotoxic agents, including ionizing radiation and chemicals used for treating cancers. The elimination of DSBs proceeds via distinctive error-free and error-prone pathways. Repair by homologous recombination is largely error-free and mediated by the RAD52 epistasis group genes. Non-homologous end joining (NHEJ) that requires the Ku heterodimer can efficiently rejoin breaks, with occasional loss or gain of DNA information. Emerging evidence has unveiled a novel DNA end-joining mechanism that is independent of Rad52 and Ku proteins. This novel pathway of DSB repair seals DNA breaks by microhomology-mediated base-pairing of DNA single strands, followed by nucleolytic trimming of DNA flaps, DNA gap filling, and DNA ligation, yielding products that are almost always associated with DNA deletion. This highly error-prone DSB repair pathway is termed microhomology-mediated end joining (MMEJ). Dissecting the mechanism of MMEJ is of great interest because of its potential to destabilize the genome through gene deletions. We have developed a Saccharomyces-based biological system to create specific double-strand breaks that are preferentially repaired by MMEJ. The availability of this MMEJ assay presents a unique opportunity to dissect the genetic requirement of this reaction. In fact, this biological system has already allowed us to identify several gene products that affect the efficiency of MMEJ. The focus of this proposal is to delineate the functions of these gene products in MMEJ and to identify additional genes that influence this process. Since the components of DSB repair are conserved from yeast to humans, the insights garnered from our research will be valuable for dissecting the equivalent process in human cells and will be of relevance to public health. [unreadable] [unreadable]